JP2008512123A5 - - Google Patents
Download PDFInfo
- Publication number
- JP2008512123A5 JP2008512123A5 JP2007531328A JP2007531328A JP2008512123A5 JP 2008512123 A5 JP2008512123 A5 JP 2008512123A5 JP 2007531328 A JP2007531328 A JP 2007531328A JP 2007531328 A JP2007531328 A JP 2007531328A JP 2008512123 A5 JP2008512123 A5 JP 2008512123A5
- Authority
- JP
- Japan
- Prior art keywords
- aqueous
- aqueous liquid
- sample
- sperm
- density
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 210000004027 cell Anatomy 0.000 claims description 78
- 239000007788 liquid Substances 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 36
- 239000008188 pellet Substances 0.000 claims description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 22
- 210000002919 epithelial cell Anatomy 0.000 claims description 18
- XTDYIOOONNVFMA-UHFFFAOYSA-N dimethyl pentanedioate Chemical compound COC(=O)CCCC(=O)OC XTDYIOOONNVFMA-UHFFFAOYSA-N 0.000 claims description 10
- 230000003196 chaotropic effect Effects 0.000 claims description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- OUWSNHWQZPEFEX-UHFFFAOYSA-N diethyl glutarate Chemical compound CCOC(=O)CCCC(=O)OCC OUWSNHWQZPEFEX-UHFFFAOYSA-N 0.000 claims description 7
- 239000012139 lysis buffer Substances 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 108010067770 Endopeptidase K Proteins 0.000 claims description 5
- 239000012062 aqueous buffer Substances 0.000 claims description 4
- JNOZGFXJZQXOSU-UHFFFAOYSA-N 1-chloro-2-methylpropan-2-ol Chemical compound CC(C)(O)CCl JNOZGFXJZQXOSU-UHFFFAOYSA-N 0.000 claims description 3
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims 1
- 108020004414 DNA Proteins 0.000 description 28
- 239000007787 solid Substances 0.000 description 8
- 239000008346 aqueous phase Substances 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 230000009089 cytolysis Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000001568 sexual effect Effects 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 4
- 239000002195 soluble material Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 239000004744 fabric Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000019419 proteases Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 210000001215 vagina Anatomy 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229940016590 sarkosyl Drugs 0.000 description 2
- 108700004121 sarkosyl Proteins 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Description
（序説）
法医学的試料から得られる遺伝物質は、性的暴行の犯人を確認するために又は罪のない容疑者を解放するために用いることができる。法医学的試料から分離した精子細胞から得られる精製されたDNAは、続いての遺伝子同一性試験において用いることができる。精子細胞DNAの遺伝子プロファイルは、既知の容疑者のそれと又は多数の有罪となった重罪犯人についての遺伝情報を有するデータベースと比較することができる。
性的暴行の場合においては、膣又は直腸をぬぐった綿棒又は精液のついた衣服が裁判分析のために犠牲者から得られる。精子細胞が試料に存在する場合には、精子細胞からDNAを分離するとともに遺伝子同一性試験に用いることができる。しかしながら、性的暴行の被害者から得られる膣をぬぐった綿棒は、典型的には、比較的少ない精子細胞と犠牲者由来の多数の上皮細胞とを含有する。結果として、精子細胞が試料中の他の細胞から最初に分離され、法医学的試料から精製されたDNAは上皮細胞DNAによる圧倒的夾雑を受けやすい。上皮細胞DNAによる夾雑は、試料からのDNA遺伝子プロファイルと容疑者又はデータベースの一人のそれとの間の一致を確立する能力を妨害する。それ故、DNA分離と分析の前に法医学的試料において他の細胞から精子細胞を分離することが望ましい。
法医学的試料において他の細胞から精子細胞を分離するために現在用いられている技術は、時間がかかると共に手間がかかり、現在処理されてない試料が滞っている。この滞っていることから、容疑者が確認されない限り、幾つかの管轄区域には試料の処理に対する方策がある。結論として、多くの処理されていない試料は最終的に捨てられ、試料に含まれる遺伝情報は決して全国的なデータベースと比較されず、それに入れられもせず、性犯罪常習者を確認し且つ逮捕する法的処置能力が低下する。
精子細胞は、典型的には、非還元条件下にプロテイナーゼＫと界面活性剤で処理することにより上皮細胞を選択的に溶解させることによって、上皮細胞を含有する法医学的試料から分離される(Gill et al. 1985)。上皮細胞溶解後、無傷の精子細胞は遠心分離によってペレット化し、溶解した上皮細胞からのDNAを含有する上清が除去される。可溶性上皮細胞DNAによる夾雑を最少にするために、可溶性上皮細胞DNAを除去する試みにおいて精子ペレットは水性緩衝液(aqueous buffer)で反復洗浄することに供される。この方法は、しばしば精子細胞の損失が生じる。
(Introduction)
Genetic material obtained from forensic samples can be used to identify perpetrators of sexual assault or to release innocent suspects. Purified DNA obtained from sperm cells isolated from forensic samples can be used in subsequent genetic identity tests. The genetic profile of sperm cell DNA can be compared to that of a known suspect or to a database having genetic information about a number of guilty felony criminals.
In the case of sexual assault, a swab or semeny clothing that wipes the vagina or rectum is obtained from the victim for court analysis. When sperm cells are present in the sample, DNA can be isolated from the sperm cells and used for genetic identity testing. However, vagina swabs obtained from victims of sexual assault typically contain relatively few sperm cells and a large number of epithelial cells from the victim. As a result, sperm cells are first isolated from other cells in the sample, and DNA purified from forensic samples is subject to overwhelming contamination by epithelial cell DNA. Contamination with epithelial cell DNA interferes with the ability to establish a match between the DNA gene profile from the sample and that of the suspect or one of the databases. It is therefore desirable to separate sperm cells from other cells in forensic samples prior to DNA separation and analysis.
The techniques currently used to separate sperm cells from other cells in forensic samples are time consuming and laborious, and currently unprocessed samples are stagnant. Because of this stagnation, some jurisdictions have strategies for sample processing unless a suspect is identified. In conclusion, many unprocessed samples are eventually discarded, and the genetic information contained in the samples is never compared to or entered into a national database, confirming and arresting sex crime addicts Legal capacity is reduced.
Sperm cells are typically separated from forensic samples containing epithelial cells by selectively lysing the epithelial cells by treatment with proteinase K and detergent under non-reducing conditions (Gill et al. 1985). After lysis of the epithelial cells, intact sperm cells are pelleted by centrifugation and the supernatant containing DNA from the lysed epithelial cells is removed. To minimize contamination with soluble epithelial cell DNA, sperm pellets are subjected to repeated washing with an aqueous buffer in an attempt to remove soluble epithelial cell DNA. This method often results in loss of sperm cells.
（発明の詳細な説明）
性的暴行の被害者から得られる法医学的試料は、典型的には、多数の上皮細胞と、存在する場合には、比較的少ない精子を含有する。遺伝子同一性試験において続いて用いられる精子細胞からDNAを得るために、結果の解釈を妨害又は複雑にすることがある水溶性材料から精子細胞、特にDNAを単離することが必要である。例えば、溶解した上皮細胞由来のDNAは、水溶液に可溶性である。
簡潔に言うと、本発明の方法は、精子細胞及び水溶液に可溶な材料を含む水性試料を、水の密度より大きいが精子細胞の密度より小さい密度を有する非水性液と接触させること、並びに、精子細胞をペレット化することを含む。適切には、精子細胞は遠心分離によってペレット化される。水溶性物質(例えば、DNA)は、水相に残り、非水相によってペレット化した精子細胞から物理的に分離される。本明細書に用いられる“精子細胞”には、無傷の精子細胞又は本質的に無傷の精子細胞と、鞭毛又は“尾部”を失った精子細胞とが含まれ得る。精子細胞は、細胞を変化させる環境条件、機械的せん断又は化学処理(例えば、プロテイナーゼＫ又は他の物質への曝露)に曝され得るが、このような細胞、特にそれらの核を保持する細胞は、本発明の範囲内に含まれる。
性的暴行の被害者からの法医学的試料は、典型的には、固体支持体、例えば、綿棒又は布(例えば、精液のついた衣服から切り取ったもの)に集められる。綿棒又は布は、試験管のような容器又は他の適切な容器に移されて、溶解緩衝液のような水溶液と接触させられ得る。実施例に記載されているように、水性緩衝液と精子細胞の回収は、固体支持体と水性緩衝液を、液体試料を通過させることを可能にする一方で固体支持体の通過を効果的に防止する機械的障壁を備えた第2容器に移し、遠心分離して、水性試料を回収することによって容易にすることができる。前記第2容器は非水性液を保持し、その結果、固体支持体からの水性試料の除去と非水性液による精子細胞のペレット化が単一の遠心分離で達成された。あるいは、非水性液による処理は同じ容器において行うことができ、固体支持体を除去する前か後のいずれかで水性試料を非水性液と接触させることによって、固体支持体を溶解緩衝液で処理してもよい。非水性液を添加した後に、容器は、遠心分離の前に固体支持体が入れられる機械的障壁を、任意に取り付けられてもよい。別のアプローチにおいては、水性試料中の精子細胞は、前記試料を非水性液と接触させる前に、遠心分離によってペレット化されてもよい。非水性液を添加すると、ペレットから水性試料が浮き、精子細胞ペレットと水性試料中の可溶性DNAとの間に障壁(即ち、非水層)が形成する。後者のアプローチは、ペレット化した精子細胞と可溶性DNAとの間の物理的分離を可能にし得るが、遠心分離の前に水性試料を非水性液と接触させる他のアプローチよりも、細胞残渣から精子細胞ペレットを効果的には分離しない。
(Detailed description of the invention)
Forensic samples obtained from victims of sexual assault typically contain a large number of epithelial cells and, if present, relatively few sperm. In order to obtain DNA from sperm cells that are subsequently used in genetic identity tests, it is necessary to isolate sperm cells, particularly DNA, from water-soluble materials that may interfere with or complicate the interpretation of the results. For example, lysed epithelial cell-derived DNA is soluble in aqueous solutions.
Briefly, the method of the present invention comprises contacting an aqueous sample comprising sperm cells and an aqueous soluble material with a non-aqueous liquid having a density greater than the density of water but less than the density of sperm cells, and Pelleting sperm cells. Suitably, sperm cells are pelleted by centrifugation. Water soluble material (eg, DNA) remains in the aqueous phase and is physically separated from sperm cells pelleted by the non-aqueous phase. As used herein, “sperm cells” can include intact or essentially intact sperm cells and sperm cells that have lost flagella or “tail”. Sperm cells can be exposed to environmental conditions that alter the cells, mechanical shear or chemical treatment (e.g., exposure to proteinase K or other substances), but such cells, particularly those that retain their nuclei, Are included within the scope of the present invention.
Forensic samples from victims of sexual assault are typically collected on a solid support, such as a cotton swab or cloth (eg, cut from semen clothing). The swab or cloth can be transferred to a container such as a test tube or other suitable container and contacted with an aqueous solution such as a lysis buffer. As described in the examples, the recovery of aqueous buffer and sperm cells effectively allows the solid support and aqueous buffer to pass through the liquid sample while effectively passing the solid support through. It can be facilitated by transferring to a second container with a mechanical barrier to prevent and centrifuging to recover the aqueous sample. The second container retained a non-aqueous liquid, so that removal of the aqueous sample from the solid support and sperm cell pelleting with the non-aqueous liquid was accomplished in a single centrifugation. Alternatively, the treatment with the non-aqueous liquid can be performed in the same container, and the solid support is treated with the lysis buffer by contacting the aqueous sample with the non-aqueous liquid either before or after removing the solid support. May be. After adding the non-aqueous liquid, the container may optionally be fitted with a mechanical barrier in which the solid support is placed prior to centrifugation. In another approach, sperm cells in an aqueous sample may be pelleted by centrifugation prior to contacting the sample with a non-aqueous liquid. When a non-aqueous liquid is added, the aqueous sample floats from the pellet and a barrier (ie, a non-aqueous layer) is formed between the sperm cell pellet and the soluble DNA in the aqueous sample. The latter approach may allow physical separation between pelleted sperm cells and soluble DNA, but from other approaches that contact aqueous samples with non-aqueous liquids prior to centrifugation, from sperm from cell debris. Does not effectively separate cell pellets.
溶解緩衝液は、適切には、プロテイナーゼＫと、サルコシル(Sarkosyl)又はSDSとを含む。任意に、溶解緩衝液は、水相の可視化を高めるのに適するいずれかの水溶性色素(例えば、FD & C イエロー)を含むことができる。材料は、上皮細胞の溶解を可能にするが精子細胞の溶解を促進しない条件下、適切な量のプロテアーゼ、例えば、プロテイナーゼＫ(270μg/ml)で処理される。精子細胞は、曝されるタンパク質が相対的に多数のジスルフィド結合を含むことから、プロテアーゼに対し相対的に抵抗性である。それ故、還元条件は、還元剤の存在は、ジスルフィド結合を破壊し、プロテアーゼで処理された精子細胞の溶解を増大させることから、差次的溶解には適さない。
プロテアーゼと界面活性剤で処理した後、溶解した上皮細胞と無傷の精子細胞とを含有する溶解物を、非水性液と接触させる。適切な非水性液には、水の密度より大きいが精子細胞の密度より小さい密度を有するいずれもの非水性液が含まれ、適切には1.00g/cm3を超える密度を有し且つ精子細胞の少なくとも一部をペレット化するのに充分小さい密度を有するあらゆる非水性液が含まれてもよい。下記の実施例に示されるように、1.29g/cm3未満の密度を有する非水性液は、続くDNAの分離、増幅及び分析のために精子細胞を回収できることがわかった。しかしながら、密度が精子細胞の少なくとも一部をペレット化できるように充分小さいことを条件として、1.29g/cm3を超える密度を有する非水性液が本発明の方法で用いることができることが構想される。本発明の方法に従って精子細胞を分離するために非水性液の適合性を評価することは、充分に当業者の能力の範囲内である。非水性液は、適切には、所望でない精子細胞の溶解を防止するように非カオトロピックである。非水性液は、好ましくは水における溶解性が相対的に低いものである。水における溶解性が低い非水性液を用いると、典型的には、より良い相分離と、上皮細胞DNAのような水溶性材料による精子細胞ペレットの夾雑の減少とが付与される。
非水性液であるジエチルグルタラート(“DEG”)、ジメチルグルタラート(“DMG”)及び1-クロロ-2-メチル-2-プロパノールを評価して、単独で又は組み合わせて用いた場合、本発明の実施において有効であることがわかった。任意に、非水性液の密度は、異なる密度を有する2つ以上の非水性液を所望の密度を得るのに有効な割合で組み合わせて用いることにより調整することができる。2つ以上の非水性液を用いる場合、それらの液体は、適切には、実質的に均一な密度の液体混合物を形成するように、実質的には相互に混和性である。
The lysis buffer suitably comprises proteinase K and Sarkosyl or SDS. Optionally, the lysis buffer can include any water-soluble dye (eg, FD & C yellow) suitable for enhancing the visualization of the aqueous phase. The material is treated with an appropriate amount of protease, eg, proteinase K (270 μg / ml) under conditions that allow lysis of epithelial cells but do not promote sperm cell lysis. Sperm cells are relatively resistant to proteases because the exposed protein contains a relatively large number of disulfide bonds. Therefore, reducing conditions are not suitable for differential lysis because the presence of a reducing agent breaks disulfide bonds and increases lysis of sperm cells treated with protease.
After treatment with protease and surfactant , a lysate containing lysed epithelial cells and intact sperm cells is contacted with a non-aqueous liquid. Suitable non-aqueous fluids include any non-aqueous fluid having a density greater than that of water but less than that of sperm cells, suitably having a density greater than 1.00 g / cm 3 and of sperm cells Any non-aqueous liquid having a density small enough to pellet at least a portion may be included. As shown in the examples below, it was found that a non-aqueous liquid having a density of less than 1.29 g / cm 3 can recover sperm cells for subsequent DNA separation, amplification and analysis. However, it is envisioned that non-aqueous liquids having a density greater than 1.29 g / cm 3 can be used in the method of the present invention, provided that the density is small enough to pellet at least some of the sperm cells. . It is well within the ability of one skilled in the art to assess the suitability of non-aqueous fluids to separate sperm cells according to the methods of the present invention. The non-aqueous liquid is suitably non-chaotropic to prevent unwanted sperm cell lysis. The non-aqueous liquid preferably has a relatively low solubility in water. Using non-aqueous liquids with low solubility in water typically provides better phase separation and reduced contamination of sperm cell pellets with water soluble materials such as epithelial cell DNA.
When non-aqueous liquids diethyl glutarate (“DEG”), dimethyl glutarate (“DMG”) and 1-chloro-2-methyl-2-propanol were evaluated and used alone or in combination, the present invention It was found to be effective in the implementation of Optionally, the density of the non-aqueous liquid can be adjusted by using two or more non-aqueous liquids having different densities in combination in proportions effective to obtain the desired density. When two or more non-aqueous liquids are used, the liquids are suitably substantially miscible with each other so as to form a liquid mixture of substantially uniform density.
水相の除去に続いて、カオトロピック剤及び還元剤を含有する溶解緩衝液を非水性液に添加しボルテックスミキサーで混合して、実質的に均一な混合物を形成することができる。実施例2に示すように、チオシアン酸グアニジン(GTC)及びジチオトレイトール(DTT)を含有する溶解緩衝液を非水相及び精子細胞ペレットと混合して、精子細胞を溶解する。実施例2は、更に、その後、精子細胞溶解物にDNAを結合することができる樹脂を添加して、他の細胞材料からDNAを精製することができることを示している。
或はまた、実施例3に記載されるように、水層と非水層双方を除去して、精子細胞ペレットを残すことができ、存在する精子細胞は、細胞を、ドデシル硫酸ナトリウム(SDS)(1% w/v)のような界面活性剤及びDTTを含む水溶液と接触させることによって溶解し、続いてフェノール:クロロホルムで抽出することができる。
具体的には、水相の除去に続いて、精子細胞ペレット及び非水相をフェノール:クロロホルム及び界面活性剤(例えば、SDS又はサルコシル)を含有する水溶液で抽出して、精子細胞DNAを遊離できることが構想される。
実施例に記載されるように、本発明の方法は、膣又は子宮頸部をぬぐった綿棒のような法医学的試料の中に含有する上皮細胞から精子細胞を分離するのに有効であることがわかった。本方法は、他の供給源から精子細胞を分離するのに有効であることが構想され、前記供給源としては、布のような精液を含有する他の固体支持体が挙げられる。本発明の方法は、精子細胞と、夾雑している赤血球又は白血球又は有核非精子細胞に由来するDNAとを含有する試料での使用に適していることが予想されるのは合理的なことである。
本発明の方法は、細胞をそれらの差次的密度に基づいて分離するのに一般的な応用性を有すること、又は選択的な溶解後、水性溶媒に可溶性の材料から他の細胞型の単離を容易にすることが予想される。本方法は、種々の細胞下の細胞小器官を分離するのに適し得ることも構想される。
以下の非限定的実施例は、純粋に具体的な説明のためのものである。
Following removal of the aqueous phase, a lysis buffer containing a chaotropic agent and a reducing agent can be added to the non-aqueous liquid and mixed with a vortex mixer to form a substantially uniform mixture. As shown in Example 2, lysis buffer containing guanidine thiocyanate (GTC) and dithiothreitol (DTT) is mixed with the non-aqueous phase and sperm cell pellet to lyse sperm cells. Example 2 further shows that DNA can then be purified from other cell materials by adding a resin capable of binding DNA to the sperm cell lysate.
Alternatively, as described in Example 3, both the aqueous and non-aqueous layers can be removed, leaving a sperm cell pellet, where the existing sperm cells can be identified as sodium dodecyl sulfate (SDS). It can be dissolved by contact with an aqueous solution containing a surfactant such as (1% w / v) and DTT , followed by extraction with phenol: chloroform.
Specifically, following removal of the aqueous phase, the sperm cell pellet and non-aqueous phase can be extracted with an aqueous solution containing phenol: chloroform and a surfactant (e.g., SDS or sarkosyl) to release sperm cell DNA. Is envisioned.
As described in the Examples, the method of the present invention may be effective in separating sperm cells from epithelial cells contained in forensic samples such as swabs that have wiped the vagina or cervix. all right. The method is envisioned to be effective in separating sperm cells from other sources, which include other solid supports containing semen such as cloth. It is reasonable that the method of the invention is expected to be suitable for use with samples containing sperm cells and DNA derived from contaminating red or white blood cells or nucleated non-sperm cells. It is.
The method of the present invention has general applicability in separating cells based on their differential density, or, after selective lysis, from other materials soluble in aqueous solvents to other cell types. It is expected to facilitate separation. It is also envisioned that the method may be suitable for separating various subcellular organelles.
The following non-limiting examples are purely illustrative.
（実施例3：界面活性剤及びフェノール:クロロホルム抽出を用いた精子細胞からDNAの分離） 実施例1の精子細胞ペレットから、最初に非水性液を除去して固まった精子細胞ペレットが残ることによってDNAを分離した。10mMトリス、pH 8.0、1mM EDTA、10mM DTT、及び1％SDSを含有する300μl溶液を精子細胞ペレットに添加し、チューブをボルテックスして、精子細胞を溶解させると共にDNAを溶解した。等容積のフェノール:クロロホルム(1:1、v/v)を添加し且つボルテックスして、タンパク質を変性させた。DNA含有水溶液を除去し、Microcon(登録商標)装置において濃縮し、10mMトリス、pH 8.0、0.1mM EDTを含有する200μlの緩衝液で洗浄し、更にまたMicrocon(登録商標)装置において約40μlまで濃縮した。精製されたDNAを清浄な容器へ移した。
Example 3: Separation of DNA from Sperm Cells Using Surfactant and Phenol: Chloroform Extraction From the sperm cell pellet of Example 1, the non-aqueous liquid is first removed to leave a solidified sperm cell pellet. DNA was isolated. A 300 μl solution containing 10 mM Tris, pH 8.0, 1 mM EDTA, 10 mM DTT, and 1% SDS was added to the sperm cell pellet, and the tube was vortexed to lyse the sperm cells and lyse the DNA. An equal volume of phenol: chloroform (1: 1, v / v) was added and vortexed to denature the protein. Remove DNA-containing aqueous solution, concentrate in Microcon® instrument, wash with 200 μl buffer containing 10 mM Tris, pH 8.0, 0.1 mM EDT, and also concentrate to approximately 40 μl in Microcon® instrument did. The purified DNA was transferred to a clean container.
Claims (28)
(a) 水性試料を1.00g/cm3より大きい密度を有する非水性液と接触させる工程であって、前記非水性液の密度が前記試料中の精子細胞の少なくとも一部をペレット化するのに充分小さい前記工程；及び
(b) 接触させた試料に、充分な時間、力を施して、水層、非水層及び精子ペレットを形成する工程；
を含む、前記方法。 A method for separating sperm cells from an aqueous sample, comprising:
(a) contacting the aqueous sample with a non-aqueous liquid having a density greater than 1.00 g / cm 3 , wherein the density of the non-aqueous liquid pellets at least some of the sperm cells in the sample. Said step small enough; and
(b) applying a force to the contacted sample for a sufficient time to form an aqueous layer, a non-aqueous layer and a sperm pellet;
Said method.
を更に含む、請求項1記載の方法。 2. The method of claim 1, further comprising (c) removing the aqueous layer.
を更に含む、請求項16記載の方法。 17. The method of claim 16, further comprising the step of (d) contacting the non-aqueous layer and sperm pellet with a chaotropic agent.
(e)工程(d)の非水層及び精子細胞ペレットを、フェノール:クロロホルムと接触させる工程
を更に含む、請求項17記載の方法。 The chaotropic agent contains a surfactant ,
18. The method of claim 17, further comprising the step of (e) contacting the non-aqueous layer and sperm cell pellet of step (d) with phenol: chloroform.
を更に含む、請求項16記載の方法。 17. The method of claim 16, further comprising (d) removing the non-aqueous layer.
を更に含む、請求項20記載の方法。 21. The method of claim 20, further comprising the step of (e) contacting the cell pellet of step (d) with an aqueous surfactant and phenol: chloroform.
a) 水性試料を1.00g/cm3より大きい密度を有する非水性液と接触させる工程であって、前記非水性液の密度が前記試料中の精子細胞の少なくとも一部をペレット化するのに充分小さい前記工程;
b) 接触させた試料を遠心分離して、水層、非水層及び精子細胞ペレットを形成する工程;c) 水層を除去する工程;
d) 非水層及び精子細胞ペレットを、カオトロピック剤及び還元剤を含む溶解緩衝液と接触させる工程; 及び
(e) 工程(d)の接触させた精子細胞ペレットからDNAを単離する工程
を含む、前記方法。 A method for isolating DNA from sperm cells in an aqueous sample containing lysed epithelial cells comprising:
a) contacting the aqueous sample with a non-aqueous liquid having a density greater than 1.00 g / cm 3 , wherein the density of the non-aqueous liquid is sufficient to pellet at least some of the sperm cells in the sample. Small said step;
b) centrifuging the contacted sample to form an aqueous layer, non-aqueous layer and sperm cell pellet; c) removing the aqueous layer;
d) contacting the non-aqueous layer and the sperm cell pellet with a lysis buffer comprising a chaotropic agent and a reducing agent; and
(e) The method comprising the step of isolating DNA from the contacted sperm cell pellet of step (d).
a) 水性試料を1.00g/cm3より大きい密度を有する非水性液と接触させる工程であって、前記非水性液の密度が前記試料中の精子細胞の少なくとも一部をペレット化するのに充分小さい前記工程;
b) 接触させた試料を遠心分離して、水層、非水層及び精子細胞ペレットを形成する工程;c) 水層及び非水層を除去する工程;
d) 精子細胞ペレットを、界面活性剤及びフェノール:クロロホルムを含む水溶液と接触させる工程; 及び
(e) 工程(d)の接触させた精子細胞ペレットからDNAを単離する工程
を含む、前記方法。 A method for isolating DNA from sperm cells in an aqueous sample containing lysed epithelial cells comprising:
a) contacting the aqueous sample with a non-aqueous liquid having a density greater than 1.00 g / cm 3 , wherein the density of the non-aqueous liquid is sufficient to pellet at least some of the sperm cells in the sample. Small said step;
b) centrifuging the contacted sample to form an aqueous layer, non-aqueous layer and sperm cell pellet; c) removing the aqueous layer and non-aqueous layer;
d) contacting the sperm cell pellet with an aqueous solution comprising a surfactant and phenol: chloroform; and
(e) The method comprising the step of isolating DNA from the contacted sperm cell pellet of step (d).
1.00g/cm3より大きい密度を有する非水性液であって、非水性液の密度が試料における精子細胞の少なくとも一部をペレット化するのに充分小さい前記非水性液を含む、前記キット。 A kit for isolating sperm cells from a sample containing sperm cells,
The kit comprising a non-aqueous liquid having a density greater than 1.00 g / cm 3, wherein the density of the non-aqueous liquid is sufficiently small to pellet at least some of the sperm cells in the sample.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10/939,105 US7320891B2 (en) | 2004-09-10 | 2004-09-10 | Methods and kits for isolating sperm cells |
| PCT/US2005/031989 WO2006031591A2 (en) | 2004-09-10 | 2005-09-07 | Methods and kits for isolating sperm cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2008512123A JP2008512123A (en) | 2008-04-24 |
| JP2008512123A5 true JP2008512123A5 (en) | 2008-10-30 |
Family
ID=36034542
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2007531328A Abandoned JP2008512123A (en) | 2004-09-10 | 2005-09-07 | Method and kit for isolating sperm cells |
Country Status (6)
| Country | Link |
|---|---|
| US (2) | US7320891B2 (en) |
| EP (1) | EP1794285A4 (en) |
| JP (1) | JP2008512123A (en) |
| AU (1) | AU2005285182A1 (en) |
| CA (1) | CA2575206A1 (en) |
| WO (1) | WO2006031591A2 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7320891B2 (en) * | 2004-09-10 | 2008-01-22 | Promega Corporation | Methods and kits for isolating sperm cells |
| BRPI0718231A2 (en) * | 2006-10-06 | 2013-11-12 | Promega Corp | METHODS AND CASES FOR INSULATING CELLS |
| EP2115123A4 (en) * | 2007-01-16 | 2010-07-07 | Applied Biosystems Llc | Selective lysis of sperm cells |
| US8993292B2 (en) | 2007-01-16 | 2015-03-31 | Applied Biosystems Llc | Methods and systems for differential extraction |
| CN103443275B (en) * | 2007-07-23 | 2016-03-16 | 应用生物系统有限责任公司 | The method of spermanucleic acid is reclaimed by forensic samples |
| US10415031B2 (en) | 2015-02-04 | 2019-09-17 | InnoGenomics Technologies, LLC | Method, apparatus and kit for human identification using polymer filter means for separation of sperm cells from biological samples that include other cell types |
| US10030241B2 (en) | 2015-03-30 | 2018-07-24 | General Electric Company | Methods and kits for capturing sperm nucleic acids |
| US9957548B2 (en) | 2015-03-30 | 2018-05-01 | General Electric Company | Methods of capturing sperm nucleic acids |
| US20210016273A1 (en) * | 2019-07-19 | 2021-01-21 | University Of Utah Research Foundation | Rapid sperm separation based on sperm morphology and motility |
| US20210284989A1 (en) * | 2020-03-13 | 2021-09-16 | Promega Corporation | Sperm cell isolation and sperm-associated dna purification |
| CN111849964A (en) * | 2020-07-03 | 2020-10-30 | 阅尔基因技术（苏州）有限公司 | Kit and method for extracting semen genome DNA |
Family Cites Families (36)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4668618A (en) * | 1981-09-14 | 1987-05-26 | Thornthwaite Jerry T | Nuclear isolation medium and procedure for separating cell nuclei |
| US5494800A (en) * | 1987-01-29 | 1996-02-27 | Cytosignet, Inc. | Analyte detection in particulate-containing samples |
| US4900685A (en) * | 1987-01-29 | 1990-02-13 | Cytosignet, Inc. | Analyte detection in particulate-containing samples |
| US5120371A (en) | 1990-11-29 | 1992-06-09 | Petroferm Inc. | Process of cleaning soldering flux and/or adhesive tape with terpenet and monobasic ester |
| US5130423A (en) * | 1990-07-13 | 1992-07-14 | Microprobe Corporation | Non-corrosive compositions and methods useful for the extraction of nucleic acids |
| US5116496A (en) * | 1991-03-19 | 1992-05-26 | Minnesota Mining And Manufacturing Company | Membrane-containing wells for microtitration and microfiltration |
| US5264184A (en) * | 1991-03-19 | 1993-11-23 | Minnesota Mining And Manufacturing Company | Device and a method for separating liquid samples |
| US5275731A (en) * | 1991-06-28 | 1994-01-04 | Jahn Karl H | Apparatus for rapidly separating blood into filtered fractions |
| CA2113218A1 (en) | 1993-10-01 | 1995-04-02 | Michael J. Oldham | Method of using lectins for contraception and prophylaxis against diseases transmittable by sexual contact and apparatus for administering lectins |
| US5766632A (en) * | 1993-10-01 | 1998-06-16 | Legere Pharmaceuticals, Ltd. | Method of using lectins for contraception |
| JP3452406B2 (en) * | 1993-10-07 | 2003-09-29 | 有限会社ケントス | Composition having performance of removing organic deposits |
| JPH10508788A (en) * | 1994-08-23 | 1998-09-02 | ベイツ，ジョン | Liquid-liquid extraction |
| US5672579A (en) * | 1995-02-06 | 1997-09-30 | Monsanto Company | Water based dimethyl ester cleaning solution |
| US5495800A (en) * | 1995-03-29 | 1996-03-05 | Cavanagh Corporation | Enhanced application printing ink hand proofing device |
| US5703193A (en) | 1996-06-03 | 1997-12-30 | Uniroyal Chemical Company, Inc. | Removal of unreacted diisocyanate monomer from polyurethane prepolymers |
| US5755987A (en) * | 1996-08-23 | 1998-05-26 | The Dow Chemical Company | Dibasic ester based phase change material compositions |
| US6027945A (en) * | 1997-01-21 | 2000-02-22 | Promega Corporation | Methods of isolating biological target materials using silica magnetic particles |
| US5916857A (en) * | 1997-11-17 | 1999-06-29 | Federal Service & Supply, Inc. | Cleaning composition for removing viscid resinous organic matter |
| GB9802973D0 (en) * | 1998-02-13 | 1998-04-08 | Du Pont Uk | Improvements in the performance of printing plates |
| US7078224B1 (en) * | 1999-05-14 | 2006-07-18 | Promega Corporation | Cell concentration and lysate clearance using paramagnetic particles |
| CA2350315C (en) * | 1998-09-10 | 2008-12-23 | Whatman Plc | Product and method for separation of a sample containing multiple sources of genetic material using a solid medium |
| US6159368A (en) * | 1998-10-29 | 2000-12-12 | The Perkin-Elmer Corporation | Multi-well microfiltration apparatus |
| GB9913556D0 (en) | 1999-06-11 | 1999-08-11 | Zeneca Ltd | Assays |
| AU6701200A (en) | 1999-08-11 | 2001-03-13 | Universiteit Gent | Isolation of cells from forensic samples for dna-typing |
| AU1778701A (en) * | 1999-11-17 | 2001-05-30 | University Of Virginia Patent Foundation | Sperm cell selection system |
| WO2001052968A1 (en) | 2000-01-24 | 2001-07-26 | Millipore Corporation | Physical separation of cells by filtration |
| US6544942B1 (en) * | 2000-04-28 | 2003-04-08 | Ecolab Inc. | Phase-separating solvent composition |
| WO2001095951A1 (en) | 2000-06-12 | 2001-12-20 | Legere Pharmaceuticals, Ltd. | Method of using lectins for contraception and prophylaxis agains t diseases transmittable by sexual contact and condom containing lectins |
| US6689615B1 (en) * | 2000-10-04 | 2004-02-10 | James Murto | Methods and devices for processing blood samples |
| WO2002048246A1 (en) * | 2000-12-16 | 2002-06-20 | Victech Co., Ltd. | Volume reducing agents for expanded polystyrene, methods and apparatus for processing expanded polystyrene using the same |
| US7060301B2 (en) * | 2001-07-13 | 2006-06-13 | Ecolab Inc. | In situ mono-or diester dicarboxylate compositions |
| EP1485402A4 (en) * | 2002-02-19 | 2006-10-11 | Choicepoint Asset Company | Selective extraction of dna from groups of cells |
| US7320891B2 (en) * | 2004-09-10 | 2008-01-22 | Promega Corporation | Methods and kits for isolating sperm cells |
| EP1929308A1 (en) * | 2005-09-01 | 2008-06-11 | Promega Corporation | Cell-based luminogenic and nonluminogenic proteasome assays |
| EP1929048A1 (en) * | 2005-09-28 | 2008-06-11 | Pierce Milwaukee, L.L.C. | Method for isolation, amplification and quantitation of ribonucleic acid |
| BRPI0718231A2 (en) * | 2006-10-06 | 2013-11-12 | Promega Corp | METHODS AND CASES FOR INSULATING CELLS |
-
2004
- 2004-09-10 US US10/939,105 patent/US7320891B2/en active Active
-
2005
- 2005-09-07 WO PCT/US2005/031989 patent/WO2006031591A2/en active Application Filing
- 2005-09-07 AU AU2005285182A patent/AU2005285182A1/en not_active Abandoned
- 2005-09-07 CA CA002575206A patent/CA2575206A1/en not_active Abandoned
- 2005-09-07 JP JP2007531328A patent/JP2008512123A/en not_active Abandoned
- 2005-09-07 EP EP05795986A patent/EP1794285A4/en not_active Withdrawn
-
2007
- 2007-11-30 US US11/947,869 patent/US20080118909A1/en not_active Abandoned
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20080118909A1 (en) | Methods and kits for isolating sperm cells | |
| EP2580323B1 (en) | Extraction of nucleic acids from wax-embedded samples | |
| JP5702717B2 (en) | Methods for isolating nucleic acids | |
| JP6204664B2 (en) | Process for parallel isolation and / or parallel purification of RNA and DNA | |
| JP5876481B2 (en) | Ultrafast nucleic acid purification method | |
| US20090202998A1 (en) | Method for the extraction of biomolecules from fixed tissues | |
| JP2010534476A (en) | Method for recovering sperm nucleic acid from forensic samples | |
| US20240175004A1 (en) | Homogeneous method to prepare sperm dna from sexual assault cases | |
| US20110300608A1 (en) | Nucleic acid isolation in preserved whole blood | |
| US8017332B2 (en) | Magnetic method for recovering nucleic acid from a mixed cell suspension | |
| JP2008512123A5 (en) | ||
| CN103890176A (en) | A method of preparing biological material | |
| EP2655617A1 (en) | Method and kit for processing wax-embedded biological samples | |
| BRPI0718231A2 (en) | METHODS AND CASES FOR INSULATING CELLS | |
| CN110945124A (en) | Method for enriching cells from a sample and subsequently isolating nucleic acids from these cells | |
| Cattaneo et al. | DNA extraction methods in forensic analysis | |
| JP2020507344A5 (en) | ||
| CN101548005A (en) | Methods and kits for isolating cells | |
| Stray et al. | Extraction of DNA from forensic biological samples for genotyping | |
| US20230142197A1 (en) | Cation exchange for sperm-associated dna purification | |
| WO2024139531A1 (en) | Kit and method for extracting dna from blood or cell sample | |
| JP2009153467A (en) | Simple method for extracting nucleic acid | |
| CN114058614A (en) | Nucleic acid extraction kit, application thereof and nucleic acid extraction method | |
| JP2016524921A (en) | How to preserve nuclei | |
| Katilius et al. | Document Title: Sperm Capture Using Aptamer Based Technology |